bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.328385; this version posted October 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Human USP18 is regulated by miRNAs via the 3’UTR, a sequence duplicated in lincRNA genes residing in chr22q11.21 Erminia Rubino1,2, Melania Cruciani1, Nicolas Tchitchek2,3, Anna Le Tortorec4, Antoine D. Rolland4, Önay Veli1, Leslie Vallet1, Giulia Gaggi1, Frédérique Michel1, Nathalie Dejucq-Rainsford4, Sandra Pellegrini1* 1 Unit of Cytokine Signaling, Institut Pasteur, INSERM U1221, Paris, France 2 École Doctorale Physiologie, Physiopathologie et Thérapeutique, ED394, Sorbonne Université, Paris, France 3 i3 research unit, Hôpital Pitié-Salpêtrière-Sorbonne Université, Paris, France 4 UMR_S1085, Irset (Institut de recherche en santé, environnement et travail), EHESP, Inserm, Univ Rennes, F- 35000 Rennes, France. Erminia Rubino : 0000-0002-7325-4736 Melania Cruciani : 0000-0002-5988-3763 Nicolas Tchitchek : 0000-0003-3307-0446 Anna Le Tortorec: 0000-0003-3306-3465 Antoine D. Rolland : 0000-0002-9758-4406 Giulia Gaggi : 0000-0002-8761-7390 Frédérique Michel : 0000-0002-3524-2232 Nathalie Dejucq-Rainsford : 0000-0002-1788-2690 Sandra Pellegrini : 0000-0001-5837-7589 * Corresponding author : Sandra Pellegrini Institut Pasteur, 25 rue du Docteur Roux 75724 Paris cedex 15, France Phone : +33 685686622 Email : [email protected] Classification : Biological Sciences, Immunology and inflammation Keywords : type I IFN; USP18; 3’UTR; lincRNA; chr22q11.21, testis. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.328385; this version posted October 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 ABSTRACT Ubiquitin-specific peptidase 18 (USP18) acts as gatekeeper of type I interferon (IFN) responses by binding to the IFN receptor subunit IFNAR2 and preventing activation of the downstream JAK/STAT pathway. In any given cell type, the level of USP18 is a key determinant of the output of interferon-stimulated transcripts. How the baseline level of USP18 is finely tuned in different cell types remains ill defined. Here we explored post-transcriptional regulation of USP18 by microRNAs (miRNAs) and identified four miRNAs (miR-24-3p, miR-191-5p, miR-423- 5p and miR-532-3p) that efficiently target USP18 through binding to the 3’UTR. Among these, three miRNAs are particularly enriched in circulating monocytes which exhibit low baseline USP18. Intriguingly, the USP18 3’UTR sequence is duplicated in human and chimpanzee genomes. In human, we found several copies of the 3’UTR that are embedded in long intergenic non-coding (linc) RNA genes residing in chr22q11.21 and exhibiting a tissue- specific expression pattern. Interestingly, one of these lincRNAs (here named linc-UR-B1) is uniquely and highly expressed in testis. RNA-seq data analyses from testicular cell subsets revealed a positive correlation between linc- UR-B1 and USP18 expression in spermatocytes and spermatids. Overall, our findings uncover a set of miRNAs and lincRNAs, which may be part of a network evolved to fine-tune baseline USP18, particularly in cell types where IFN responsiveness needs to be tightly controlled. SIGNIFICANT STATEMENT USP18 is a non-redundant negative feedback regulator of type I IFN signaling and a key determinant of cell responsiveness to IFN. How baseline USP18 is set in different human cell types is ill defined. We identified three microRNAs that restrain USP18 level notably in primary monocytes through binding the 3’UTR. We found several copies of the USP18 3’UTR embedded in long intergenic non-coding (linc) RNAs which reside in a complex region of human chromosome 22. These lincRNAs are expressed in a tissue-specific manner. We describe one lincRNA expressed only in testis, and most notably in germ cells. Correlative analyses suggest that microRNAs and lincRNAs may form a network controlling baseline USP18 and IFN responsiveness. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.328385; this version posted October 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 INTRODUCTION Ubiquitin-specific peptidase 18 (USP18) is an interferon-stimulated gene (ISG) exerting a specific and non- redundant role in the negative regulation of type I IFN (here IFN) responses (1). USP18 is recruited to the IFNAR2 receptor subunit, interferes with JAK1/TYK2 activation, and attenuates STAT-mediated ISGs induction. USP18 is also an isopeptidase (de-ISGylase) that non-redundantly cleaves the ubiquitin-like ISG15 from protein conjugates (2-4). Deficiency in USP18 causes a severe type I interferonopathy resulting in perinatal death with serious brain malformations due to spontaneous microglia activation, which most likely results from unrestrained response to constitutive IFNb (5). Indeed, high baseline USP18 maintains microglia quiescence and prevents sub- threshold activation (6, 7). In mouse models of VSV and LCMV infection, it was shown that the ability of CD169+ spleen macrophages and dendritic cells to present viral antigens and elicit an innate and adaptive immunity relies on the expression of Usp18 (8, 9). Altogether, these findings point to USP18 as a key determinant of cell responsiveness to IFN, including in the context of constitutive low IFNb levels. We and others have shown that in humans, but not in mice, free ISG15 sustains the level of USP18, by preventing its proteasomal degradation by the S-phase kinase-associated protein 2 (SKP2) (10-12). Yet, little is known on how baseline USP18 levels are set in different cell lineages. MicroRNAs (miRNAs) act as fine-tuners of gene expression and protein output. Each gene target can be regulated by numerous miRNAs, which typically bind to specific sites in 3’ untranslated regions (3’UTRs) of mRNAs, influencing mRNA stability and translation. Hence, the amount of target varies from cell to cell depending on the identity and the expression level of each miRNA (13). MiRNAs operate in different physiological and pathological processes (14) and also participate to the complex network that regulates immune cell development, function and response to stimuli (15). As for many immune pathways, miRNAs have been shown to regulate the IFN system both at the level of production and response. Hence, some of the key players of the type I IFN signaling pathway are under the control of miRNAs (16), but no miRNA targeting USP18 has been described so far. Here, we investigated whether miRNAs contribute to tune down baseline USP18 in a cell-context specific manner. We identified four miRNAs that negatively regulate USP18 through binding the 3’UTR. We also report on the presence of several copies of the USP18 3’UTR in the human genome. These duplications reside on chr22q11.21 and are embedded in long intergenic non-coding RNA (lincRNA) genes, whose expression is tissue- specific. The remarkable cell type-specific expression of one such lincRNA in testicular germ cells and its positive correlation with USP18 raises the possibility that the USP18 3’UTR not only acts as a cis-regulatory sequence targeted by miRNAs, but also acts in trans when embedded in lincRNA molecules expressed in particular cell types. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.328385; this version posted October 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 4 RESULTS The 3’UTR of human USP18 is targeted by at least four miRNAs Using the bioinformatic prediction programs miRWalk and RNAhybrid, we identified 27 miRNAs predicted to target with high score the 580 nucleotide (nt)-long 3’UTR of the human USP18 mRNA (Fig. 1A). USP18 is basally detected in most human tissues, albeit at variable levels (Fig. S1). We therefore analyzed the expression of the 27 miRNA candidates in the 90 cell types of the FANTOM5 dataset (17). Fourteen miRNAs were found to be expressed at > 50 CPM (counts per million mapped reads) in at least one cell type (Fig. 1A). We next tested the functional impact of these 14 miRNAs on USP18. For this, we expressed in HeLa S3 cells each miRNA as mimic and measured the level of USP18 mRNA by qPCR. As shown in Fig. 1B, seven miRNAs led to a reduction of endogenous USP18, four of them exhibiting a stronger effect. Consistently, this also resulted in lower USP18 protein levels, at least for those four miRNAs with stronger effects at the RNA level (Fig. 1C). In order to validate the binding of each miRNA to the predicted site on USP18 3’UTR (Fig. 1D, E), this latter sequence was subcloned downstream of the Renilla luciferase gene in the psiCHECK2 vector and the impact of the four miRNAs on luciferase was measured in transfected HeLa S3 cells. As shown in Fig. 1F, miR-191-5p, miR-24-3p, miR-423-5p and miR-532-3p, expressed as mimics, significantly decreased luciferase activity with respect to the control mimic. Moreover, the effect of each mimic was abrogated when a 5 nt mutation was introduced in the seed-matched sequence of the predicted binding site on the 3’UTR (Fig. 1F). Altogether these data demonstrate that the USP18 3’UTR sequence can be directly targeted by miR-191-5p, miR-24-3p, miR-423- 5p and miR-532-3p, with an effect on USP18 abundance. To determine in which cell type(s) this post-transcriptional control may operate, we performed a principal component analysis (PCA) based on the expression of the seven active miRNAs identified above in the 90 cell types (Fig. 2A). The analysis revealed a different distribution of immune vs non-immune cells, with the first three PC vectors explaining 75% of the total variance.